Balazs Sarkadi 1,2, Katalin Meszaros 2,3,4, Ildiko Krencz 5, Sara Zakarias 1, Kinga Nemeth 1, Gabor Barna 5, Anna Sebestyen 5, Judit Papay 5, Katalin Borka 7, Zoltan Hujber 5, Miklos Toth 1, Peter Igaz 1, Chinopoulos Christos 6, Attila Patocs 2,3,4
1. 2ndDepartment of Medicine, Semmelweis University, Budapest, Hungary
2.“Lendület” Hereditary Endocrine Tumours Research Group, HAS-SE, Budapest, Hungary
3. Department of Laboratory Medicine, Semmelweis University, Budapest, Hungary
4. Bionics Innovation Center, Budapest, Hungary
5. 1st Department of Pathology and Experimental Cancer, Semmelweis University, Budapest, Hungary
6. Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary
7. 2nd Department of Pathology, Semmelweis University, Budapest, Hungary
INTRODUCTION: Pheochromocytomas and paragangliomas (Pheo/PGL) exhibit a unique metabolic phenotype due to mutations of genes encoding the succinate dehydrogenase (SDH) subunits. Via pharmacological treatments and gene silencing, we aimed to assess the metabolic and viability consequences of SDH inhibition in order to identify an in-vitro model for SDH impairment. Furthermore, we evaluated the expression of glutaminase-1 (GLS-1) in hereditary Pheo/PGL tissues.
MATERIALS AND METHODS: GLS-1 expression was assessed on 15 Pheo/PGL tumor tissue blocks obtained from 9 patients with hereditary Pheo/PGL. SDHB silencing with siRNA was accomplished in PC12 cells. The pharmacological inhibition of SDH was achieved with itaconate or atpenin A5 on PC12, HeLa and H295R cells. Liquid chromatography-mass spectrometry was used to assess the metabolic profiles of the cells. Data were normalized to DNA concentrations. Cellular viability was determined with alamarBlue.
RESULTS: The expression of GLS-1 positively correlated with malignancy in Pheo/PGL tumors. All SDH inhibitory method increased significantly the succinate/fumarate ratio in all studied cell lines. Cell viability of PC12 cells significantly increased after SDHB siRNA transfection and itaconate treatment. Itaconate significantly decreased HeLa and H295R cells’ viability. Atpenin significantly increased HeLa cell line’s viability and decreased viability of H295R cells, while no effect on PC12 cells was observed.
Different metabolic phenotypes were observed depending on the inhibition method and cell line. In PC12 cells glutamate concentrations were significantly decreased without lactate accumulation after itaconate or SDHB knockdown. HeLa and H295R cells accumulated both lactate and glutamate upon itaconate treatment. Atpenin treatment didn’t yield cell specific results: all cell lines exhibited significantly increased lactate and decreased glutamate concentrations.
CONCLUSION: Itaconate mimics the SDHB knockdown phenotype in PC12 cells. The results of SDH inhibition are cell line and method specific. Based on the increased GLS-1 expression, targeting glutamate/glutamine metabolism might yield a novel therapeutic option for malignant Pheo/PGL.
Doctoral School: Clinical Medicine
Program: Hormonal Regulations
Supervisor: Attila Patócs
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